1. Field of the Invention
This invention relates to a fermentation method to produce succinic acid, and more particularly this invention relates to a method for creating bacterial strains capable of utilizing a myriad of sugars to produce succinic acid as a major fermentation product.
2. Background of the Invention
Carboxylic acids hold promise as potential precursors for numerous chemicals. For example, succinic acid can serve as a feedstock for such plastic precursors as 1,4-butanediol (BDO), tetrahydrofuran, and gamma-butyrolactone. New products derived from succinic acid are under development, with the most notable of these being polyester which is made by linking succinic acid and BDO. Generally, esters of succinic acids have the potential of being new, "green" solvents that can supplant more harmful solvents. In total, succinic acid could serve as a precursor for millions of pounds of chemicals annually at a total market value of over $1 billion. Along with succinic acid, other 4-carbon dicarboxylic acids, such as malic acid, and fumaric acid also have feedstock potential.
The production of these carboxylic acids from renewable feedstocks (in this case through fermentation processes) is an avenue to supplant the more energy intensive methods of deriving such acids from nonrenewable sources.
Succinate is an intermediate for anaerobic fermentations by propionate-producing bacteria but those processes result in low yields and concentrations.
Anaerobic rumen bacteria, such as Bacteroides ruminicola and Bacteroides amylophilus also produce succinate. However, rumen organisms are characteristically unstable in fermentation processes.
It has long been known that a mixture of acids are produced from E. coli fermentation, as elaborated in Stokes, J. L. 1949 "Fermentation of glucose by suspensions of Escherichia coli" J. Bacteriol. 57:147-158. However, for each mole of glucose fermented, only 1.2 moles of formic acid, 0.1-0.2 moles of lactic acid, and 0.3-0.4 moles of succinic acid are produced. As such, efforts to produce carboxylic acids fermentatively have resulted in relatively large amounts of growth substrates, such as glucose, not being converted to desired product.
Some bacteria, such as A. succiniciproducens, utilized in fermentation processes as outlined in U.S. Pat. No. 5,143,834 to Glassner et al., naturally produce succinic acid in moderate titers up to only about 35-40 grams per liter (g/L). The A. succiniciproducens host strain has been shown to be not highly osmotolerant in that it does not tolerate high concentrations of salts and is further inhibited by moderate concentrations of product. Lastly, A. succiniciproducens presents handling problems in that as an obligate anaerobe, procedures using the organism must be done in the absence of oxygen. Also, medium preparation for the inoculum requires the addition of tryptophan and also requires the addition of a solution of cysteine, HCl, and sodium sulfide which contains corrosive and toxic H.sub.2 S.
Previous efforts by the inventors to produce succinic acid has resulted in the isolation and utilization of a mutant bacterium. The mutant, available as ATCC accession number 202021, is the subject of U.S. patent application Ser. No. 08/556,805 to the instant Assignee. U.S. application Ser. No. 08/556,805, incorporated herein by reference, teaches a succinic acid-producing bacterial strain (AFP 111) which spontaneously mutates from its precursor. The mutant is able to grow fermentatively on glucose to produce succinic acid in high yields, while its precursor is unable to do so. However, an obvious drawback to utilizing this method of succinic acid production is its limitation to a single mutant organism.
None of the prior attempts outlined supra have resulted in a process for feeding a myriad of feedstocks to several different metabolizing organisms to produce high quantities of succinic acid. Such flexibility in types of nutrients and organisms utilized would result in exploiting the lower costs of many feedstocks, such as corn stover, logging slash, and other lignocellulosic feedstocks, and the higher product tolerances and robust metabolisms of certain organisms.
A need exists in the art for a fermentation process whereby a myriad of feedstocks can be fed to several different metabolizing organisms to produce large quantities of succinic acid. The process should utilize low cost nutrients, and perhaps different organisms to facilitate fermentation in various environments. The process should also yield molar ratios of approximately 1:1 product-to-growth substrate.